Associations in cell type-specific hydroxymethylation and transcriptional alterations of pediatric central nervous system tumors.

Although intratumoral heterogeneity has been established in pediatric central nervous system tumors, epigenomic alterations at the cell type level have largely remained unresolved. To identify cell type-specific alterations to cytosine modifications in pediatric central nervous system tumors, we utilize a multi-omic approach that integrated bulk DNA cytosine modification data (methylation and hydroxymethylation) with both bulk and single-cell RNA-sequencing data. We demonstrate a large reduction in the scope of significantly differentially modified cytosines in tumors when accounting for tumor cell type composition. In the progenitor-like cell types of tumors, we identify a preponderance differential Cytosine-phosphate-Guanine site hydroxymethylation rather than methylation. Genes with differential hydroxymethylation, like histone deacetylase 4 and insulin-like growth factor 1 receptor, are associated with cell type-specific changes in gene expression in tumors. Our results highlight the importance of epigenomic alterations in the progenitor-like cell types and its role in cell type-specific transcriptional regulation in pediatric central nervous system tumors.

Immunological shifts during early-stage Parkinson's disease identified with DNA methylation data on longitudinally collected blood samples.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the United States. Decades before motor symptoms manifest, non-motor symptoms such as hyposmia and rapid eye movement (REM) sleep behavior disorder are highly predictive of PD. Previous immune profiling studies have identified alterations to the proportions of immune cells in the blood of clinically defined PD patients. However, it remains unclear if these phenotypes manifest before the clinical diagnosis of PD. We utilized longitudinal DNA methylation (DNAm) microarray data from the Parkinson's Progression Marker's Initiative (PPMI) to perform immune profiling in clinically defined PD and prodromal PD patients (Prod). We identified previously reported changes in neutrophil, monocyte, and T cell numbers in PD patients. Additionally, we noted previously unrecognized decreases in the naive B cell compartment in the defined PD and Prod patient group. Over time, we observed the proportion of innate immune cells in PD blood increased, but the proportion of adaptive immune cells decreased. We identified decreases in T and B cell subsets associated with REM sleep disturbances and early cognitive decline. Lastly, we identified increases in B memory cells associated with both genetic (LRRK2 genotype) and infectious (cytomegalovirus seropositivity) risk factors of PD. Our analysis shows that the peripheral immune system is dynamic as the disease progresses. The study provides a platform to understand how and when peripheral immune alterations occur in PD and whether intervention at particular stages may be therapeutically advantageous.

Matched analysis of detailed peripheral blood and tumor immune microenvironment profiles in bladder cancer.

Background: Bladder cancer and therapy responses hinge on immune profiles in the tumor microenvironment (TME) and blood, yet studies linking tumor-infiltrating immune cells to peripheral immune profiles are limited. Methods: DNA methylation cytometry quantified TME and matched peripheral blood immune cell proportions. With tumor immune profile data as the input, subjects were grouped by immune infiltration status and consensus clustering. Results: Immune hot and cold groups had different immune compositions in the TME but not in circulating blood. Two clusters of patients identified with consensus clustering had different immune compositions not only in the TME but also in blood. Conclusion: Detailed immune profiling via methylation cytometry reveals the significance of understanding tumor and systemic immune relationships in cancer patients.

Bladder cancer and treatment outcomes depend on the immune profiles in the tumor and blood. Our study, using DNA methylation cytometry, measured immune cell proportions in both areas. Patients were grouped based on immune status and consensus clustering. Results showed distinct immune compositions in the tumor, but not in blood, for hot and cold groups. Consensus clustering revealed two patient clusters with differing immune compositions in both tumor and blood. This detailed immune profiling highlights the importance of understanding the complex interplay between tumor and systemic immunity in bladder cancer patients.

Tumor microenvironment deconvolution identifies cell-type-independent aberrant DNA methylation and gene expression in prostate cancer.

BACKGROUND: Among men, prostate cancer (PCa) is the second most common cancer and the second leading cause of cancer death. Etiologic factors associated with both prostate carcinogenesis and somatic alterations in tumors are incompletely understood. While genetic variants associated with PCa have been identified, epigenetic alterations in PCa are relatively understudied. To date, DNA methylation (DNAm) and gene expression (GE) in PCa have been investigated; however, these studies did not correct for cell-type proportions of the tumor microenvironment (TME), which could confound results. METHODS: The data (GSE183040) consisted of DNAm and GE data from both tumor and adjacent non-tumor prostate tissue of 56 patients who underwent radical prostatectomies prior to any treatment. This study builds upon previous studies that examined methylation patterns and GE in PCa patients by using a novel tumor deconvolution approach to identify and correct for cell-type proportions of the TME in its epigenome-wide association study (EWAS) and differential expression analysis (DEA). RESULTS: The inclusion of cell-type proportions in EWASs and DEAs reduced the scope of significant alterations associated with PCa. We identified 2,093 significantly differentially methylated CpGs (DMC), and 51 genes associated with PCa, including PCA3, SPINK1, and AMACR. CONCLUSIONS: This work illustrates the importance of correcting for cell types of the TME when performing EWASs and DEAs on PCa samples, and establishes a more confounding-adverse methodology. We identified a more tumor-cell-specific set of altered genes and epigenetic marks that can be further investigated as potential biomarkers of disease or potential therapeutic targets.

Detailed immune profiling in pediatric Crohn's disease using methylation cytometry.

DNA methylation has been extensively utilized to study epigenetic patterns across many diseases as well as to deconvolve blood cell type proportions. This study builds upon previous studies examining methylation patterns in paediatric patients with varying stages of Crohn's disease to extend the immune profiling of these patients using a novel deconvolution approach. Compared with control subjects, we observed significantly decreased levels of CD4 memory and naive, CD8 naive, and natural killer cells and elevated neutrophil levels in Crohn's disease. In addition, Crohn's patients had a significantly elevated neutrophil-to-lymphocyte ratio. Using an epigenome-wide association approach and adjusting for potential confounders, including cell type, we observed 397 differentially methylated CpG (DMC) sites associated with Crohn's disease. The top genetic pathway associated with the DMCs was the regulation of arginine metabolic processes which are involved in the regulation of T cells.

Potential to Enhance Large Scale Molecular Assessments of Skin Photoaging through Virtual Inference of Spatial Transcriptomics from Routine Staining.

The advent of spatial transcriptomics technologies has heralded a renaissance in research to advance our understanding of the spatial cellular and transcriptional heterogeneity within tissues. Spatial transcriptomics allows investigation of the interplay between cells, molecular pathways, and the surrounding tissue architecture and can help elucidate developmental trajectories, disease pathogenesis, and various niches in the tumor microenvironment. Photoaging is the histological and molecular skin damage resulting from chronic/acute sun exposure and is a major risk factor for skin cancer. Spatial transcriptomics technologies hold promise for improving the reliability of evaluating photoaging and developing new therapeutics. Challenges to current methods include limited focus on dermal elastosis variations and reliance on self-reported measures, which can introduce subjectivity and inconsistency. Spatial transcriptomics offers an opportunity to assess photoaging objectively and reproducibly in studies of carcinogenesis and discern the effectiveness of therapies that intervene in photoaging and preventing cancer. Evaluation of distinct histological architectures using highly-multiplexed spatial technologies can identify specific cell lineages that have been understudied due to their location beyond the depth of UV penetration. However, the cost and interpatient variability using state-of-the-art assays such as the 10x Genomics Spatial Transcriptomics assays limits the scope and scale of large-scale molecular epidemiologic studies. Here, we investigate the inference of spatial transcriptomics information from routine hematoxylin and eosin-stained (H&E) tissue slides. We employed the Visium CytAssist spatial transcriptomics assay to analyze over 18,000 genes at a 50-micron resolution for four patients from a cohort of 261 skin specimens collected adjacent to surgical resection sites for basal cell and squamous cell keratinocyte tumors. The spatial transcriptomics data was co-registered with 40x resolution whole slide imaging (WSI) information. We developed machine learning models that achieved a macro-averaged median AUC and F1 score of 0.80 and 0.61 and Spearman coefficient of 0.60 in inferring transcriptomic profiles across the slides, and accurately captured biological pathways across various tissue architectures.

Deciphering the role of immune cell composition in epigenetic age acceleration: Insights from cell-type deconvolution applied to human blood epigenetic clocks.

Aging is a significant risk factor for various human disorders, and DNA methylation clocks have emerged as powerful tools for estimating biological age and predicting health-related outcomes. Methylation data from blood DNA has been a focus of more recently developed DNA methylation clocks. However, the impact of immune cell composition on epigenetic age acceleration (EAA) remains unclear as only some clocks incorporate partial cell type composition information when analyzing EAA. We investigated associations of 12 immune cell types measured by cell-type deconvolution with EAA predicted by six widely-used DNA methylation clocks in data from >10,000 blood samples. We observed significant associations of immune cell composition with EAA for all six clocks tested. Across the clocks, nine or more of the 12 cell types tested exhibited significant associations with EAA. Higher memory lymphocyte subtype proportions were associated with increased EAA, and naïve lymphocyte subtypes were associated with decreased EAA. To demonstrate the potential confounding of EAA by immune cell composition, we applied EAA in rheumatoid arthritis. Our research maps immune cell type contributions to EAA in human blood and offers opportunities to adjust for immune cell composition in EAA studies to a significantly more granular level. Understanding associations of EAA with immune profiles has implications for the interpretation of epigenetic age and its relevance in aging and disease research. Our detailed map of immune cell type contributions serves as a resource for studies utilizing epigenetic clocks across diverse research fields, including aging-related diseases, precision medicine, and therapeutic interventions.

Spatial Omics Driven Crossmodal Pretraining Applied to Graph-based Deep Learning for Cancer Pathology Analysis.

Graph-based deep learning has shown great promise in cancer histopathology image analysis by contextualizing complex morphology and structure across whole slide images to make high quality downstream outcome predictions (ex: prognostication). These methods rely on informative representations (i.e., embeddings) of image patches comprising larger slides, which are used as node attributes in slide graphs. Spatial omics data, including spatial transcriptomics, is a novel paradigm offering a wealth of detailed information. Pairing this data with corresponding histological imaging localized at 50-micron resolution, may facilitate the development of algorithms which better appreciate the morphological and molecular underpinnings of carcinogenesis. Here, we explore the utility of leveraging spatial transcriptomics data with a contrastive crossmodal pretraining mechanism to generate deep learning models that can extract molecular and histological information for graph-based learning tasks. Performance on cancer staging, lymph node metastasis prediction, survival prediction, and tissue clustering analyses indicate that the proposed methods bring improvement to graph based deep learning models for histopathological slides compared to leveraging histological information from existing schemes, demonstrating the promise of mining spatial omics data to enhance deep learning for pathology workflows.

Molecular subtypes of high-grade serous ovarian cancer across racial groups and gene expression platforms.

Introduction: High-grade serous carcinoma (HGSC) gene expression subtypes are associated with differential survival. We characterized HGSC gene expression in Black individuals and considered whether gene expression differences by race may contribute to poorer HGSC survival among Black versus non-Hispanic White individuals. Methods: We included newly generated RNA-Seq data from Black and White individuals, and array-based genotyping data from four existing studies of White and Japanese individuals. We assigned subtypes using K-means clustering. Cluster- and dataset-specific gene expression patterns were summarized by moderated t-scores. We compared cluster-specific gene expression patterns across datasets by calculating the correlation between the summarized vectors of moderated t-scores. Following mapping to The Cancer Genome Atlas (TCGA)-derived HGSC subtypes, we used Cox proportional hazards models to estimate subtype-specific survival by dataset. Results: Cluster-specific gene expression was similar across gene expression platforms. Comparing the Black study population to the White and Japanese study populations, the immunoreactive subtype was more common (39% versus 23%-28%) and the differentiated subtype less common (7% versus 22%-31%). Patterns of subtype-specific survival were similar between the Black and White populations with RNA-Seq data; compared to mesenchymal cases, the risk of death was similar for proliferative and differentiated cases and suggestively lower for immunoreactive cases (Black population HR=0.79 [0.55, 1.13], White population HR=0.86 [0.62, 1.19]). Conclusions: A single, platform-agnostic pipeline can be used to assign HGSC gene expression subtypes. While the observed prevalence of HGSC subtypes varied by race, subtype-specific survival was similar.

Feasibility of Inferring Spatial Transcriptomics from Single-Cell Histological Patterns for Studying Colon Cancer Tumor Heterogeneity.

Background: Spatial transcriptomics involves studying the spatial organization of gene expression within tissues, offering insights into the molecular diversity of tumors. While spatial gene expression is commonly amalgamated from 1-10 cells across 50-micron spots, recent methods have demonstrated the capability to disaggregate this information at subspot resolution by leveraging both expression and histological patterns. However, elucidating such information from histology alone presents a significant challenge but if solved can better permit spatial molecular analysis at cellular resolution for instances where Visium data is not available, reducing study costs. This study explores integrating single-cell histological and transcriptomic data to infer spatial mRNA expression patterns in whole slide images collected from a cohort of stage pT3 colorectal cancer patients. A cell graph neural network algorithm was developed to align histological information extracted from detected cells with single cell RNA patterns through optimal transport methods, facilitating the analysis of cellular groupings and gene relationships. This approach leveraged spot-level expression as an intermediary to co-map histological and transcriptomic information at the single-cell level. Results: Our study demonstrated that single-cell transcriptional heterogeneity within a spot could be predicted from histological markers extracted from cells detected within a spot. Furthermore, our model exhibited proficiency in delineating overarching gene expression patterns across whole-slide images. This approach compared favorably to traditional patch-based computer vision methods as well as other methods which did not incorporate single cell expression during the model fitting procedures. Topological nuances of single-cell expression within a Visium spot were preserved using the developed methodology. Conclusion: This innovative approach augments the resolution of spatial molecular assays utilizing histology as a sole input through synergistic co-mapping of histological and transcriptomic datasets at the single-cell level, anchored by spatial transcriptomics. While initial results are promising, they warrant rigorous validation. This includes collaborating with pathologists for precise spatial identification of distinct cell types and utilizing sophisticated assays, such as Xenium, to attain deeper subcellular insights.

The Overlooked Role of Specimen Preparation in Bolstering Deep Learning-Enhanced Spatial Transcriptomics Workflows.

The application of deep learning methods to spatial transcriptomics has shown promise in unraveling the complex relationships between gene expression patterns and tissue architecture as they pertain to various pathological conditions. Deep learning methods that can infer gene expression patterns directly from tissue histomorphology can expand the capability to discern spatial molecular markers within tissue slides. However, current methods utilizing these techniques are plagued by substantial variability in tissue preparation and characteristics, which can hinder the broader adoption of these tools. Furthermore, training deep learning models using spatial transcriptomics on small study cohorts remains a costly endeavor. Necessitating novel tissue preparation processes enhance assay reliability, resolution, and scalability. This study investigated the impact of an enhanced specimen processing workflow for facilitating a deep learning-based spatial transcriptomics assessment. The enhanced workflow leveraged the flexibility of the Visium CytAssist assay to permit automated H&E staining (e.g., Leica Bond) of tissue slides, whole-slide imaging at 40x-resolution, and multiplexing of tissue sections from multiple patients within individual capture areas for spatial transcriptomics profiling. Using a cohort of thirteen pT3 stage colorectal cancer (CRC) patients, we compared the efficacy of deep learning models trained on slide prepared using an enhanced workflow as compared to the traditional workflow which leverages manual tissue staining and standard imaging of tissue slides. Leveraging Inceptionv3 neural networks, we aimed to predict gene expression patterns across matched serial tissue sections, each stemming from a distinct workflow but aligned based on persistent histological structures. Findings indicate that the enhanced workflow considerably outperformed the traditional spatial transcriptomics workflow. Gene expression profiles predicted from enhanced tissue slides also yielded expression patterns more topologically consistent with the ground truth. This led to enhanced statistical precision in pinpointing biomarkers associated with distinct spatial structures. These insights can potentially elevate diagnostic and prognostic biomarker detection by broadening the range of spatial molecular markers linked to metastasis and recurrence. Future endeavors will further explore these findings to enrich our comprehension of various diseases and uncover molecular pathways with greater nuance. Combining deep learning with spatial transcriptomics provides a compelling avenue to enrich our understanding of tumor biology and improve clinical outcomes. For results of the highest fidelity, however, effective specimen processing is crucial, and fostering collaboration between histotechnicians, pathologists, and genomics specialists is essential to herald this new era in spatial transcriptomics-driven cancer research.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude.

BACKGROUND: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear. METHODS: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points. RESULTS: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N). CONCLUSIONS: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.

Genome-Scale Methylation Analysis Identifies Immune Profiles and Age Acceleration Associations with Bladder Cancer Outcomes.

BACKGROUND: Immune profiles have been associated with bladder cancer outcomes and may have clinical applications for prognosis. However, associations of detailed immune cell subtypes with patient outcomes remain underexplored and may contribute crucial prognostic information for better managing bladder cancer recurrence and survival. METHODS: Bladder cancer case peripheral blood DNA methylation was measured using the Illumina HumanMethylationEPIC array. Extended cell-type deconvolution quantified 12 immune cell-type proportions, including memory, naïve T and B cells, and granulocyte subtypes. DNA methylation clocks determined biological age. Cox proportional hazards models tested associations of immune cell profiles and age acceleration with bladder cancer outcomes. The partDSA algorithm discriminated 10-year overall survival groups from clinical variables and immune cell profiles, and a semi-supervised recursively partitioned mixture model (SS-RPMM) with DNA methylation data was applied to identify a classifier for 10-year overall survival. RESULTS: Higher CD8T memory cell proportions were associated with better overall survival [HR = 0.95, 95% confidence interval (CI) = 0.93-0.98], while higher neutrophil-to-lymphocyte ratio (HR = 1.36, 95% CI = 1.23-1.50), CD8T naïve (HR = 1.21, 95% CI = 1.04-1.41), neutrophil (HR = 1.04, 95% CI = 1.03-1.06) proportions, and age acceleration (HR = 1.06, 95% CI = 1.03-1.08) were associated with worse overall survival in patient with bladder cancer. partDSA and SS-RPMM classified five groups of subjects with significant differences in overall survival. CONCLUSIONS: We identified associations between immune cell subtypes and age acceleration with bladder cancer outcomes. IMPACT: The findings of this study suggest that bladder cancer outcomes are associated with specific methylation-derived immune cell-type proportions and age acceleration, and these factors could be potential prognostic biomarkers.

Hierarchical deconvolution for extensive cell type resolution in the human brain using DNA methylation.

Introduction: The human brain comprises heterogeneous cell types whose composition can be altered with physiological and pathological conditions. New approaches to discern the diversity and distribution of brain cells associated with neurological conditions would significantly advance the study of brain-related pathophysiology and neuroscience. Unlike single-nuclei approaches, DNA methylation-based deconvolution does not require special sample handling or processing, is cost-effective, and easily scales to large study designs. Existing DNA methylation-based methods for brain cell deconvolution are limited in the number of cell types deconvolved. Methods: Using DNA methylation profiles of the top cell-type-specific differentially methylated CpGs, we employed a hierarchical modeling approach to deconvolve GABAergic neurons, glutamatergic neurons, astrocytes, microglial cells, oligodendrocytes, endothelial cells, and stromal cells. Results: We demonstrate the utility of our method by applying it to data on normal tissues from various brain regions and in aging and diseased tissues, including Alzheimer's disease, autism, Huntington's disease, epilepsy, and schizophrenia. Discussion: We expect that the ability to determine the cellular composition in the brain using only DNA from bulk samples will accelerate understanding brain cell type composition and cell-type-specific epigenetic states in normal and diseased brain tissues.

Normal gastric tissue Helicobacter pylori infection is associated with epigenetic age acceleration, increased mitotic tick rate, tissue cell composition, and Natural Killer cell methylation alterations.

Background: Gastric adenocarcinomas are a leading cause of global mortality, associated with chronic infection with Helicobacter pylori. The mechanisms by which infection with H. pylori contributes to carcinogenesis are not well understood. Recent studies from subjects with and without gastric cancer have identified significant DNA methylation alterations in normal gastric mucosa associated with H. pylori infection and gastric cancer risk. Here we further investigated DNA methylation alterations in normal gastric mucosa in gastric cancer cases (n = 42) and control subjects (n = 42) with H. pylori infection data. We assessed tissue cell type composition, DNA methylation alterations within cell populations, epigenetic aging, and repetitive element methylation. Results: In normal gastric mucosa of both gastric cancer cases and control subjects, we observed increased epigenetic age acceleration associated with H. pylori infection. We also observed an increased mitotic tick rate associated with H. pylori infection in both gastric cancer cases and controls. Significant differences in immune cell populations associated with H. pylori infection in normal tissue from cancer cases and controls were identified using DNA methylation cell type deconvolution. We also found natural killer cell-specific methylation alterations in normal mucosa from gastric cancer patients with H. pylori infection. Conclusions: Our findings from normal gastric mucosa provide insight into underlying cellular composition and epigenetic aspects of H. pylori associated gastric cancer etiology.

Assessment of emerging pretraining strategies in interpretable multimodal deep learning for cancer prognostication.

BACKGROUND: Deep learning models can infer cancer patient prognosis from molecular and anatomic pathology information. Recent studies that leveraged information from complementary multimodal data improved prognostication, further illustrating the potential utility of such methods. However, current approaches: 1) do not comprehensively leverage biological and histomorphological relationships and 2) make use of emerging strategies to "pretrain" models (i.e., train models on a slightly orthogonal dataset/modeling objective) which may aid prognostication by reducing the amount of information required for achieving optimal performance. In addition, model interpretation is crucial for facilitating the clinical adoption of deep learning methods by fostering practitioner understanding and trust in the technology. METHODS: Here, we develop an interpretable multimodal modeling framework that combines DNA methylation, gene expression, and histopathology (i.e., tissue slides) data, and we compare performance of crossmodal pretraining, contrastive learning, and transfer learning versus the standard procedure. RESULTS: Our models outperform the existing state-of-the-art method (average 11.54% C-index increase), and baseline clinically driven models (average 11.7% C-index increase). Model interpretations elucidate consideration of biologically meaningful factors in making prognosis predictions. DISCUSSION: Our results demonstrate that the selection of pretraining strategies is crucial for obtaining highly accurate prognostication models, even more so than devising an innovative model architecture, and further emphasize the all-important role of the tumor microenvironment on disease progression.

Calculating detection limits and uncertainty of reference-based deconvolution of whole-blood DNA methylation data.

DNA methylation (DNAm)-based cell mixture deconvolution (CMD) has become a quintessential part of epigenome-wide association studies where DNAm is profiled in heterogeneous tissue types. Despite being introduced over a decade ago, detection limits, which represent the smallest fraction of a cell type in a mixed biospecimen that can be reliably detected, have yet to be determined in the context of DNAm-based CMD. Moreover, there has been little attention given to approaches for quantifying the uncertainty associated with DNAm-based CMD. Here, analytical frameworks for determining both cell-specific limits of detection and quantification of uncertainty associated with DNAm-based CMD are described. This work may contribute to improved rigor, reproducibility and replicability of epigenome-wide association studies involving CMD.

Down Syndrome Altered Cell Composition in Blood, Brain, and Buccal Swab Samples Profiled by DNA-Methylation-Based Cell-Type Deconvolution.

Down syndrome (DS) is a genetic disorder caused by an extra copy of chromosome 21 that presents developmental dysfunction and intellectual disability. To better understand the cellular changes associated with DS, we investigated the cell composition in blood, brain, and buccal swab samples from DS patients and controls using DNA methylation-based cell-type deconvolution. We used genome-scale DNA methylation data from Illumina HumanMethylation450k and HumanMethylationEPIC arrays to profile cell composition and trace fetal lineage cells in blood samples (DS N = 46; control N = 1469), brain samples from various regions (DS N = 71; control N = 101), and buccal swab samples (DS N = 10; control N = 10). In early development, the number of cells from the fetal lineage in the blood is drastically lower in DS patients (Δ = 17.5%), indicating an epigenetically dysregulated maturation process for DS patients. Across sample types, we observed significant alterations in relative cell-type proportions for DS subjects compared with the controls. Cell-type proportion alterations were present in samples from early development and adulthood. Our findings provide insight into DS cellular biology and suggest potential cellular interventional targets for DS.

Identification of Spatial Proteomic Signatures of Colon Tumor Metastasis: A Digital Spatial Profiling Approach.

Over 150,000 Americans are diagnosed with colorectal cancer (CRC) every year, and annually >50,000 individuals are estimated to die of CRC, necessitating improvements in screening, prognostication, disease management, and therapeutic options. CRC tumors are removed en bloc with surrounding vasculature and lymphatics. Examination of regional lymph nodes at the time of surgical resection is essential for prognostication. Developing alternative approaches to indirectly assess recurrence risk would have utility in cases where lymph node yield is incomplete or inadequate. Spatially dependent, immune cell-specific (eg, tumor-infiltrating lymphocytes), proteomic, and transcriptomic expression patterns inside and around the tumor-the tumor immune microenvironment-can predict nodal/distant metastasis and probe the coordinated immune response from the primary tumor site. The comprehensive characterization of tumor-infiltrating lymphocytes and other immune infiltrates is possible using highly multiplexed spatial omics technologies, such as the GeoMX Digital Spatial Profiler. In this study, machine learning and differential co-expression analyses helped identify biomarkers from Digital Spatial Profiler-assayed protein expression patterns inside, at the invasive margin, and away from the tumor, associated with extracellular matrix remodeling (eg, granzyme B and fibronectin), immune suppression (eg, forkhead box P3), exhaustion and cytotoxicity (eg, CD8), Programmed death ligand 1-expressing dendritic cells, and neutrophil proliferation, among other concomitant alterations. Further investigation of these biomarkers may reveal independent risk factors of CRC metastasis that can be formulated into low-cost, widely available assays.

HiTAIC: hierarchical tumor artificial intelligence classifier traces tissue of origin and tumor type in primary and metastasized tumors using DNA methylation.

Human cancers are heterogenous by their cell composition and origination site. Cancer metastasis generates the conundrum of the unknown origin of migrated tumor cells. Tracing tissue of origin and tumor type in primary and metastasized cancer is vital for clinical significance. DNA methylation alterations play a crucial role in carcinogenesis and mark cell fate differentiation, thus can be used to trace tumor tissue of origin. In this study, we employed a novel tumor-type-specific hierarchical model using genome-scale DNA methylation data to develop a multilayer perceptron model, HiTAIC, to trace tissue of origin and tumor type in 27 cancers from 23 tissue sites in data from 7735 tumors with high resolution, accuracy, and specificity. In tracing primary cancer origin, HiTAIC accuracy was 99% in the test set and 93% in the external validation data set. Metastatic cancers were identified with a 96% accuracy in the external data set. HiTAIC is a user-friendly web-based application through https://sites.dartmouth.edu/salaslabhitaic/. In conclusion, we developed HiTAIC, a DNA methylation-based algorithm, to trace tumor tissue of origin in primary and metastasized cancers. The high accuracy and resolution of tumor tracing using HiTAIC holds promise for clinical assistance in identifying cancer of unknown origin.